Summary Higher order chromatin organization is perturbed in cancer cells. It is hypothesized that these perturbations are closely linked to carcinogenesis and cancer phenotype. However, a fulsome picture of how chromatin organization contributes to cancer phenotype still eludes us. This question is unlikely to be answered by looking at just a handful of cells or cell types, since there is a tremendous variety in cancer phenotype. It is also unlikely that looking at aggregate or population data will answer these questions, since there is heterogeneity in chromosome organization from one cell to the next. Current tools to study chromatin organization have proven unequal to this challenging ask, either because they can only look at a few cells at a time (microscopy based techniques) or they only provide fragmented snapshots with no spatial context (chromosome conformation capture based assays). We propose the development of a novel biochemical assay, a molecular ruler, that combines the strength of microscopy techniques (multicolor labeling and spatial context) with the high-throughput, scalable, single-molecule approach of new generation chromosome conformation capture assays. Molecular rulers will label chromosomes with barcoded DNA probes and generate DNA records that encode the absolute distance between labeled sites in their length. We will first develop and calibrate the molecular ruler technology on a flat DNA origami substrate (Aim 1.1) and then test our ability to accurately reconstruct the geometry of a 3D object with single-molecule resolution, in this case, an asymmetric wireframe DNA origami tetrahedron (Aim 1.2). Next, we will develop the molecular technology on a population of fixed K562 (human chronic myelogenous leukemia) cells to reconstruct aggregate chromosome geometry (Aim 2.1) and then introduce single molecule barcodes to enable single molecule reconstruction of chromosome geometry (Aim 2.2). This approach will give us the ability to assay chromosome geometry in a wide variety and number of cell states and types, at single cell resolution, while also providing critical spatial context that will help computer models reconstruct chromosome geometry in single cells. This ability is critical in answering questions about the role played by chromosome architecture in carcinogenesis and the cancer phenotype.